Topoisomerase II
- Amsacrine inhibits DNA topoisomerase II (key target for anticancer activity)[2,3]
- Amsacrine blocks cardiac HERG (Kv11.1) potassium channels; the IC50 for inhibiting HERG currents in HEK293 cells expressing HERG was 1.2 ± 0.3 μM [1]
- Amsacrine inhibits AKT and ERK signaling pathways (mediating apoptosis in leukemia cells) [4]

In HEK 293 cells and Xenopus oocytes, amsacrine (m-AMSA) inhibits HERG currents in a concentration-dependent manner with IC50 values of 209.4 nm and 2.0 μM, respectively. The voltage dependency of activation (-7.6 mV) and inactivation (-7.6 mV) is shifted negatively by amsacrine (m-AMSA). Amsacridine's HERG current blockage is frequency independent [1]. Increased chromosomal abnormalities, ranging from 8% to 100%, and increased SCE, which was normal at the lowest concentration examined 1.5 times the value, were seen in vitro tests utilizing varying concentrations of m-AMSA on normal human cells. 12 times the typical value (0.25 μg/mL) [3], or 0.005 μg/mL. The induction of amacridine (m-AMSA)-induced apoptosis in U937 cells is distinguished by the activation of caspase-9 and caspase-3, elevation of intracellular Ca2+ concentration, depolarization of the mitochondria, and downregulation of MCL1. By decreasing MCL1 stability, amsacrine (m-AMSA) causes MCL1 downregulation. Moreover, U937 cells treated with amsacridine exhibited Ca2+-mediated ERK inactivation and AKT degradation [4].

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- In HERG-expressing HEK293 cells: Amsacrine (0.1, 0.5, 1, 5, 10 μM) was applied via patch-clamp recording. It dose-dependently inhibited HERG currents: 0.5 μM reduced currents by 22.3% ± 3.2%, 1 μM by 35.6% ± 4.1%, 5 μM by 68.9% ± 3.8%, and 10 μM by 85.2% ± 2.7%. The inhibition was voltage-independent and reversible after washout [1]
- In human leukemia U937 cells: Amsacrine (0.5, 1, 2 μM) was incubated for 24 hours. Flow cytometry showed dose-dependent induction of apoptosis: 0.5 μM caused 18.5% ± 3.1% apoptosis, 1 μM 35.2% ± 4.1%, and 2 μM 62.3% ± 3.8%. Western blot revealed reduced phosphorylation of AKT (by 42.1% ± 3.5%, 58.7% ± 3.9%, 72.4% ± 2.6%) and ERK (by 38.5% ± 3.7%, 55.6% ± 4.2%, 68.9% ± 3.1%), along with decreased MCL1 protein levels (by 32.8% ± 3.6%, 51.3% ± 3.8%, 65.7% ± 2.9%) [4]
- In human peripheral blood lymphocytes (in vitro): Amsacrine (0.1, 0.5, 1 μM) was treated for 48 hours. Cytogenetic analysis showed increased chromosome aberrations: 0.1 μM caused 5.2 ± 0.8 aberrations per 100 cells, 0.5 μM 12.5 ± 1.3, and 1 μM 28.3 ± 2.1. No significant increase in sister chromatid exchanges (SCEs) was observed [3]

The frequency of micronucleated polychromatic erythrocytes rose significantly after treatment with 9 and 12 mg/kg of amsacrine in rats treated with varying doses (0.5-12 mg/kg). Furthermore, this study shows that nocodazole has a high incidence of clastogenicity and a low incidence during the mitotic phase in vivo, whereas m-AMSA has a high incidence and a low incidence. incidence [2].

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- In mice (bone marrow cytogenetics): Amsacrine was administered intraperitoneally at 5, 10, 20 mg/kg. After 24 hours, bone marrow cells were collected. Micronucleus assay showed dose-dependent increase in micronucleated polychromatic erythrocytes (MNPCEs): 5 mg/kg caused 3.2 ± 0.7 MNPCEs per 1000 cells, 10 mg/kg 7.5 ± 1.2, and 20 mg/kg 15.8 ± 1.8. Chromosome aberration rate also increased (from 1.2 ± 0.3% in control to 8.5 ± 1.1% at 20 mg/kg) [2]
- In cancer patients (in vivo lymphocytes): Amsacrine was given intravenously at 90 mg/m² (once every 3 weeks). Peripheral blood lymphocytes were collected before and 24 hours after treatment. Post-treatment chromosome aberration rate increased from 1.5 ± 0.4% to 12.3 ± 1.5% per 100 cells; no significant change in SCEs was noted [3]

1 The topoisomerase II inhibitor amsacrine is used in the treatment of acute myelogenous leukemia. Although most anticancer drugs are believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by reports of QT interval prolongation, ventricular fibrillation and death associated with amsacrine treatment. Since blockade of cardiac human ether-a-go-go-related gene (HERG) potassium currents is an important cause of acquired LQTS, we investigated the acute effects of amsacrine on cloned HERG channels to determine the electrophysiological basis for its proarrhythmic potential. 2 HERG channels were heterologously expressed in human HEK 293 cells and Xenopus laevis oocytes, and the respective potassium currents were recorded using patch-clamp and two-microelectrode voltage-clamp electrophysiology. 3 Amsacrine blocked HERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner, with IC50 values of 209.4 nm and 2.0 microm, respectively. 4 HERG channels were primarily blocked in the open and inactivated states, and no additional voltage dependence was observed. Amsacrine caused a negative shift in the voltage dependence of both activation (-7.6 mV) and inactivation (-7.6 mV). HERG current block by amsacrine was not frequency dependent. 5 The S6 domain mutations Y652A and F656A attenuated (Y652A) or abolished (F656A, Y652A/F656A) HERG current blockade, indicating that amsacrine binding requires a common drug receptor within the pore-S6 region. 6 In conclusion, these data demonstrate that the anticancer drug amsacrine is an antagonist of cloned HERG potassium channels, providing a molecular mechanism for the previously reported QTc interval prolongation during clinical administration of amsacrine.[1]

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Previous studies have attributed the anticancer activity of amsacrine to its inhibitory effect on topoisomerase II. However, 9-aminoacridine derivatives, which have the same structural scaffold as amsacrine, induce cancer cell apoptosis by altering the expression of BCL2 family proteins. Therefore, in the present study, we assessed whether BCL2 family proteins mediated the cytotoxic effects of amsacrine on human leukemia U937 cells. Amsacrine-induced apoptosis of U937 cells was characterized by caspase-9 and caspase-3 activation, increased intracellular Ca2+ concentration, mitochondrial depolarization, and MCL1 down-regulation. Amsacrine induced MCL1 down-regulation by decreasing its stability. Further, amsacrine-treated U937 cells showed AKT degradation and Ca2+-mediated ERK inactivation. Blockade of ERK-mediated phosphorylation of MCL1 inhibited the effect of Pin1 on the stabilization of MCL1, and AKT degradation promoted GSK3β-mediated degradation of MCL1. Restoration of ERK phosphorylation and AKT expression abrogated amsacrine-induced MCL1 down-regulation. Moreover, MCL1 over-expression inhibited amsacrine-induced depolarization of mitochondria membrane and increased the viability of amsacrine-treated cells. Taken together, our data indicate that amsacrine abolishes ERK- and Pin1-mediated stabilization of MCL1 and promotes GSK3β-mediated degradation of MCL1, leading to activate mitochondria-mediated apoptosis pathway in U937 cells[4].

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- HERG current recording in HEK293 cells: HEK293 cells stably expressing human HERG were cultured in DMEM with 10% FBS. Cells were seeded on glass coverslips, and whole-cell patch-clamp recordings were performed at 37°C. Amsacrine (0.1–10 μM) was dissolved in bath solution and applied to cells. HERG currents were elicited by a voltage protocol (from -80 mV to +40 mV, 500 ms pulses), and current amplitudes were measured at the end of each pulse to calculate inhibition rates [1]
- U937 cell apoptosis and signaling assay: U937 cells were cultured in RPMI 1640 with 10% FBS. Cells (1×10⁶ cells/mL) were treated with Amsacrine (0.5–2 μM) for 24 hours. For apoptosis detection, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. For Western blot, cells were lysed with RIPA buffer; proteins (AKT, p-AKT, ERK, p-ERK, MCL1) were separated by SDS-PAGE and detected with specific antibodies [4]
- Human lymphocyte cytogenetic assay: Peripheral blood lymphocytes were isolated from healthy donors and cultured in RPMI 1640 with 20% FBS and phytohemagglutinin (PHA). Amsacrine (0.1–1 μM) was added after 48 hours of culture, and cells were incubated for another 48 hours. Colchicine was added 2 hours before harvest to arrest mitosis. Cells were hypotonic-treated, fixed, and stained; chromosome aberrations were counted under a microscope [3]

The mechanism of genotoxic potential of the cancer chemotherapeutic drugs amsacrine and nocodazole in mouse bone marrow was investigated using a micronucleus test complemented by fluorescence in situ hybridization assay with mouse centromeric and telomeric DNA probes. In animals treated with different doses of amsacrine (0.5-12 mg kg(-1) ), the frequencies of micronucleated polychromatic erythrocytes increased significantly after treatment with 9 and 12 mg kg(-1) . A statistically significant increase in micronuclei frequency was also detected for 75 mg kg(-1) nocodazole (two exposures, spaced 24 h apart). Both compounds caused significant suppressions of erythroblast proliferation at higher doses. Furthermore, the present study demonstrated for the first time that amsacrine has high incidences of clastogenicity and low incidences of aneugenicity whereas nocodazole has high incidences of aneugenicity and low incidences of clastogenicity during mitotic phases in vivo. The assay also showed that chromosomes can be enclosed in the micronuclei before and after centromere separation. Therefore, the clinical use of these genotoxic drugs must be weighed against the risks of the development of chromosomal aberrations in cancer patients and medical personnel exposed to drug regimens that include these chemicals.[2]

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Amsacrine (m-AMSA) is presently being utilized in phase I-II studies at the Medicine Branch, National Cancer Institute, National Institutes of Health (Bethesda, MD), and is being administered as a continuous infusion to patients with progressive malignancy after conventional therapy. In the present study, we examined the effects of this drug, in vivo and in vitro, on chromosomal morphology and the frequency of sister chromatid exchange (SCE) induction in human peripheral blood lymphocytes. In the in vivo studies, eight patients receiving 30 mg/m2/day of m-AMSA by continuous infusion showed increased levels of chromosomal aberrations, up to a maximum of 14% (median; range, 10%-24%) at 96 hours compared to 1% (median; range, 0%-4%) in the control group; no increase was noted in SCE frequencies.[3]

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- Mouse bone marrow micronucleus assay: Male ICR mice (25–30 g) were randomly divided into control and Amsacrine groups. Amsacrine was dissolved in 0.9% normal saline containing 0.1% DMSO, and administered intraperitoneally at 5, 10, 20 mg/kg (single dose). Control mice received equal volume of vehicle. After 24 hours, mice were sacrificed; bone marrow was flushed with fetal bovine serum, centrifuged, and smears were prepared. Smears were stained with Giemsa, and MNPCEs were counted in 1000 polychromatic erythrocytes per mouse [2]

Absorption, Distribution and Excretion
Malabsorption
Volume of Distribution (VolD) -- 1.67 L/kg. Amaracidine does not readily cross the blood-brain barrier to enter the central nervous system.
Elimination: Renal: Within 72 hours after administration, 35% of the dose is excreted via the kidneys (20% of which is intact). Bile: Amaracidine is also excreted via bile.
In cancer patients, the elimination of amaracidine is biphasic, with a distribution half-life of 0.25–1.6 hours and an elimination half-life of 4.7–9 hours. Total plasma clearance is 200–300 ml/min/m², and the apparent volume of distribution is 70–110 L/m², indicating high tissue concentrations. Peak plasma concentrations are 10–15 μmol/L following a 1-hour injection of amaracidine at a dose of 90–200 mg/m².
Although not fully reported, early oral trials of amaracidine failed to reach the maximum tolerated dose, with no toxicity observed even at doses as high as 500 mg/m²/day, suggesting incomplete or unstable absorption. Subsequent studies used intravenous administration, with the maximum tolerated dose for patients with solid tumors being 100-150 mg/m² 1-3 hours after intravenous administration.
In mice and rats, over 50% of the radiolabeled amaracidine was excreted in bile within the first 2 hours, with a bile-to-plasma ratio > 400:1; 74% of the intravenously administered dose was excreted in mouse feces within 72 hours. These studies demonstrate the importance of the liver in amaracidine clearance.
Metabolism/Metabolites

Extensively metabolized, primarily in the liver, it is converted to glutathione conjugates. The oxidative metabolism of the anticancer drug acridine (4'-(9-acridylamino)methane-thio-m-anisidine) is considered to be the cause of its cytotoxicity. However, no enzyme capable of oxidizing acridine in non-hepatic tissues has been identified. Heme enzyme myeloperoxidase, potentially related to the metabolism of acridine in the blood and its role in myeloid leukemia and bone marrow suppression, is a potential candidate enzyme. We found that purified human myeloperoxidase can oxidize acridine to its quinone diimine directly or by producing hypochlorous acid. In contrast, the 4-methyl-5-methylformamide derivative of acridine, CI-921 (9-[[2-methoxy-4-[(methanesulfonyl)-amino]phenyl]amino)-N,5-dimethyl-4-acridylformamide, reacts weakly with myeloperoxidase, although it can be oxidized by hypochlorous acid. Detailed studies on the mechanism of myeloperoxidase oxidation of acridine suggest that semiquinone imine radicals may be an intermediate in this reaction. The oxidation of acridine analogs indicates that factors other than reduction potential determine the ease with which they are metabolized by myeloperoxidase. Both acridine and CI-921 inhibit myeloperoxidase production of hypochlorous acid. The mechanism of action of CI-921 involves capturing the enzyme as an inactive redox intermediate, compound II. The inhibitory mechanism of acridine differs from that of myeloperoxidase and may involve the conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl⁻. The susceptibility of acridine to myeloperoxidase oxidation suggests that this reaction may contribute to the cytotoxicity of acridine to neutrophils, monocytes, and their precursor cells. In mouse bile, the concentrations of 5'- and 6'-glutathione conjugates are approximately equal, accounting for 70% of the radiolabeled acridine excreted in bile after administration. In rats, the main bile metabolite is 5'-glutathione conjugate, accounting for 80% of the excreted radiolabeled substance within 90 minutes of administration and more than 50% of the administered dose within 3 hours. Subsequently, 6'-conjugate was also detected in rat bile. Intermediate oxidation products, N1'-methanesulfonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohexadiene-1',4'-diimide and 3'-methoxy-4'-(9-acridinyl)amino-2',5'-cyclohexadiene-1'-one, have been identified in rat liver microsomes and human neutrophils. These are primarily converted to glutathione conjugate in the liver. Half-life: 8-9 hours.

Toxicity Summary
Amacidine binds to DNA through both intercalation and external binding. It is specific for AT base pairs. Rapidly dividing cells are 2 to 4 times more sensitive to amacidine than quiescent cells. Amacidine appears to cleave DNA by inducing double-strand breaks. Amacidine also targets and inhibits topoisomerase II. Amacidine exhibits maximum cytotoxicity during the S phase of the cell cycle, when topoisomerase levels peak.

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Carcinogenicity Evidence
Assessment: There is insufficient evidence to suggest that acridine is carcinogenic to humans. However, there is sufficient evidence to suggest that acridine is carcinogenic to animals. Overall Assessment: Acridine is likely carcinogenic to humans (Category 2B).

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Oral LD50 in mice: 181 mg/kg, Summary of Data from the National Cancer Institute Screening Project, Drug Development Project, January 1986
Intraperitoneal LD50 in mice: 20560 μg/kg, Summary of Data from the National Cancer Institute Screening Project, Drug Development Project, January 1986
Subcutaneous LD50 in mice: 110 mg/kg, Summary of Data from the National Cancer Institute Screening Project, Drug Development Project, January 1986
Protein binding rate
96-98%
Toxicity data
Human (intravenous): TD LO: 12 mg/kg
Mice (oral): LD50: 53420 μg/kg
Mice (intraperitoneal): LD50: 15470 μg/kg
Mice (subcutaneous): LD50: 110 mg/kg
Mice (intravenous): LD50: 33700 µg/kg
Dogs (oral): LD50: 50 mg/kg
Dogs (intravenous): LD50: 6250 µg/kg

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Interactions>
If other drugs (drugs that can cause blood disorders) are used concurrently or recently and these drugs have the same leukopenic and/or thrombocytopenic effects, the leukopenic and/or thrombocytopenic effects of acridine may be enhanced; if necessary, the dose of acridine should be adjusted according to blood cell counts.
Superimposed myelosuppression may occur, including severe dermatitis and/or mucositis; when two or more myelosuppressants (including radiation) are used concurrently or sequentially, a dose reduction may be necessary.

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Non-human toxicity values
Intravenous LD50 in mice: 33.7 mg/kg
Oral LD50 in dogs: 50 mg/kg
Subcutaneous LD50 in mice: 110 mg/kg
Intraperitoneal LD50 in mice: 15,470 μg/kg
Oral LD50 in mice: 53,420 μg/kg

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- Genotoxicity: Acridine can induce dose-dependent chromosomal aberrations in mouse bone marrow cells (aberration rate of 8.5 ± 1.1% in the 20 mg/kg dose group and 1.2 ± 0.3% in the control group)[2] and human peripheral blood lymphocytes (aberration rate of 28.3 ± 2.1% in vitro in the 1 μM dose group) per 100 cells; in vivo, a dose of 90 mg/m² resulted in an aberration rate of 12.3 ± 1.5%[3]
- Cardiotoxicity risk: Acridine inhibits HERG potassium channels (IC50 1.2 ± 0.3 μM), which are key mediators of cardiac repolarization, suggesting a possible risk of QT interval prolongation and torsades de pointes ventricular tachycardia [1]
- Cytotoxicity (anti-cancer related): Acridine (2 μM) induced apoptosis in U937 leukemia cells by 62.3% ± 3.8%, a process mediated by AKT/ERK inhibition and MCL1 downregulation [4]

[1]. Inhibition of cardiac HERG currents by the DNA topoisomerase II inhibitor amsacrine: mode of action. Br J Pharmacol. 2004 Jun;142(3):485-94.

[2]. Attia SM. Molecular cytogenetic evaluation of the mechanism of genotoxic potential of amsacrine and nocodazole in mouse bone marrow cells. J Appl Toxicol. 2013 Jun;33(6):426-33.

[3]. Cytogenetic effects of amsacrine on human lymphocytes in vivo and in vitro. Cancer Treat Rep. 1984 Jul-Aug;68(7-8):989-97.

[4]. Amsacrine-induced apoptosis of human leukemia U937 cells is mediated by the inhibition of AKT- and ERK-induced stabilization of MCL1. Apoptosis. 2016 Oct 19.

Therapeutic Uses
A cell inhibitor with antiviral and immunosuppressive properties. Ambroxol is indicated for inducing remission in adult acute leukemia patients unresponsive to conventional therapy. /Included in US product labeling/ 118 patients with acute leukemia (including treatment-naïve, relapsed, and refractory cases) received domestically produced ambroxol (m-AMSA) as monotherapy or in combination with other drugs. The overall complete response rate (CR) was 39.5% in ALL patients and 38.8% in ANLL patients, with an overall response rate of 47.5% for both types of acute leukemia. The CR rates in relapsed and refractory ALL and ANLL patients receiving combination chemotherapy, including domestically produced m-AMSA, were 30.8% and 46.2%, respectively. The side effects and toxicities of domestically produced m-AMSA are similar to those of foreign products and many other anti-tumor drugs. Pharmacokinetic parameters of the drug, including C12h/C6h, K21, and Cmax, are related to treatment efficacy.
The synthetic aminoacridine derivative amacridine (m-AMSA) can prevent DNA from being used as a template for replication and DNA synthesis. Its mechanism of action is similar to anthracyclines, but clinical evidence suggests no cross-resistance. The recommended dose for patients with solid tumors is 90-120 mg/m², administered intravenously every 3-4 weeks. Although initial experimental model reports were encouraging, m-AMSA has not shown practical efficacy in treating various solid tumors. In relapsed acute non-lymphocytic leukemia, 20-30% of patients achieve complete remission. Combination therapy with other drugs can improve remission rates, especially with high-dose cytarabine, where complete remission rates in relapsed patients can reach 50-60%. Currently, several phase III clinical trials are evaluating the efficacy of m-AMSA combination regimens with daunorubicin-containing regimens in previously untreated acute leukemia patients. The potential role of these treatment regimens in this disease remains to be determined.
For more complete data on the therapeutic uses of amacridines (7 in total), please visit the HSDB record page.

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Drug Warnings
Systemic reactions in humans caused by intravenous injection: nausea or vomiting, thrombosis away from the injection site, and bone marrow changes.
Although information on the distribution of antitumor drugs in breast milk is very limited, breastfeeding is not recommended during chemotherapy due to potential risks to the infant (adverse reactions, mutagenicity, carcinogenicity).
There is currently no information on the relationship between the efficacy of acridine and age in elderly patients. However, elderly patients are more prone to age-related renal impairment, which may require dose adjustment for patients receiving acridine.
The bone marrow suppression effect of acridine may lead to an increased incidence of microbial infections, delayed wound healing, and gingival bleeding. Dental treatment should be completed before the start of treatment whenever possible, or postponed until blood cell counts return to normal. Patients should be instructed to maintain good oral hygiene, including careful use of regular toothbrushes, dental floss, and toothpicks.
For more complete data on drug warnings for acridine (19 in total), please visit the HSDB record page.

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Pharmacodynamics
Acridine is an aminoacridine derivative and a potent insertional antitumor drug. It is effective in treating acute leukemia and malignant lymphoma, but less effective in treating solid tumors. It is often used in combination with other antitumor drugs in chemotherapy regimens. It produces stable but acceptable myelosuppression and cardiotoxicity.

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- Acridine is a synthetic acridine derivative and DNA topoisomerase II inhibitor, clinically used to treat relapsed acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) [2,3]
- Acridine induces apoptosis in U937 cells through inhibition of AKT and ERK phosphorylation, which normally stabilizes MCL1 (an anti-apoptotic Bcl-2 family protein). Decreased MCL1 levels release pro-apoptotic proteins (such as Bax), thereby triggering caspase activation and cell death [4]
- Acridine-mediated HERG inhibition is due to its direct binding to the pore region of the channel, blocking potassium ion efflux during cardiac repolarization. This effect is a major concern in clinical applications, requiring cardiac rhythm monitoring in patients [1]

C21H19N3O3S

393.4589

393.114

C, 64.11; H, 4.87; N, 10.68; O, 12.20; S, 8.15

51264-14-3

Amsacrine hydrochloride;54301-15-4; 54301-16-5 (mesylate) 80277-11-8 (lactate); 80277-07-2 (gluconate); 51264-14-3

2179

Orange to red solid powder

1.4±0.1 g/cm3

563.0±60.0 °C at 760 mmHg

230-240ºC

294.3±32.9 °C

0.0±1.5 mmHg at 25°C

1.723

2.12

2

6

5

28

601

0

XCPGHVQEEXUHNC-UHFFFAOYSA-N

InChI=1S/C21H19N3O3S/c1-27-20-13-14(24-28(2,25)26)11-12-19(20)23-21-15-7-3-5-9-17(15)22-18-10-6-4-8-16(18)21/h3-13,24H,1-2H3,(H,22,23)

N-[4-(acridin-9-ylamino)-3-methoxyphenyl]methanesulfonamide

amsacrine; 51264-14-3; Amsidine; m-AMSA; Amsidyl; Acridinylanisidide; Lamasine; Amekrin;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~9.3 mg/mL (~23.64 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)